Description:

CLIP-Cell™ Block (bromothenylcytosine, BTC) is a non-fluorescent compound that blocks the reactivity of the CLIP-tag™ in vitro and inside or on the surface of living cells. It can be used to generate inactive controls in live cell labeling experiments performed with CLIP-tag fusion proteins. BTC reacts with CLIP-tag irreversibly, inactivating it for subsequent labeling steps. 

The CLIP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest. CLIP-tag is a protein tag based on human O⁶-alkylguanine-DNAalkyltransferase (hAGT). CLIP-tag substrates are derivatives of benzylcytosine (BC). In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the reactive cysteine of CLIP-tag forming a stable thioether bond.  Although CLIP-tag is based on the same protein as SNAP-tag®, the benzylcytosine substrates form a separate class of substrates, different from the benzylguanine substrates recognized by SNAP-tag. CLIP-tag and SNAP-tag can be used for orthogonal and complementary labeling of two proteins simultaneously in the same cells. 

There are two steps to using this system: subcloning and expression of the protein of interest as a CLIP-tag fusion, and labeling of the fusion with the CLIP-tag substrate of choice. The use of BTC during the labeling of fusion proteins with CLIP-Cell substrates is described in this document.

Figure 1: Structure of CLIP-Cell Block (MW 286.1)

Notes:

Storage: CLIP-Cell Block should be stored at -20°C (long term) or at 4°C (short term). With proper storage at -20°C, CLIP-Cell Block is stable for at least three years dry or for three months when dissolved in DMSO.For troubleshooting please refer to the instructions supplied with CLIP-Cell products as appropriate.Please note that there is a constant turnover and resynthesis of proteins in the cell. After having blocked all existing CLIP-tag fusion proteins within the cell, new CLIP-tag fusion protein molecules may be synthesized in the meantime and may get labeled during incubation with a fluorescent CLIP-tag substrate. This will give the impression that the blocking was ineffective. In order to minimize these effects of protein synthesis and protein transport, cells may have to be treated with cycloheximide and incubation with the fluorescent CLIP-tag substrate may have to be performed at 4°C.

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